Embryonic thermal conditioning and post-hatch heat challenge in broiler chicks reveal changes in hypothalamic expression of genes related to appetite, thermoregulation, and stress modulation

Embryonic heat conditioning (EHC; exposing the chick embryo to elevated temperatures transiently during incubation) has been associated with greater stress resiliency later in life, although mechanisms are unclear but likely involve the hypothalamus. The objective of this study was to determine the effects of an acute heat challenge at day 4 post-hatch on the transcriptome of several brain nuclei associated with thermal regulation, stress, and appetite, including the paraventricular nucleus of the hypothalamus (PVN), pre-optic anterior/hypothalamic area (POAH), and the nucleus of the hippocampal commissure (nCPa), in broilers that were subjected to control or EHC. The nuclei were collected by punch biopsy at 3 time points relative to the start of heat challenge (0, 2 and 12 hours), total RNA was isolated, and RNA-sequencing was performed. Transcript abundance was quantified and differentially expressed genes (DEG) identified, and gene ontology analyses were performed. In the nCPa, 469 DEGs were identified across the 3 timepoints. There were 0 DEGs at hour 0, 2 at hour 2, and 467 at hour 12. The gene ontology analysis on nCPa hour 12 samples revealed enrichment of 5 biological processes including mitochondrial electron transport, mitochondrial respiratory chain complex 1 assembly, synaptic vesicle lumen acidification, protein export from nucleus, and aerobic respiration. Most of these genes were down-regulated suggesting that these processes were less functional in EHC chicks. In the POAH, total 18 DEGs were identified, with 0 at hour 0, 18 at hour 2, and 0 at hour 12. Fewer differences were observed in the PVN, with only 4 DEGs identified. All 4 were up-regulated in the EHC group, with 2 being involved in hypothalamic thermal responses: vasoactive intestinal peptide transporter 1 (VIPR1) and caprin family member 2 (CAPRIN2). We also compared expression across time. There were no differences identified in the POAH or PVN. In the nCPA, no differences were detected for hour 2 vs. hour 0, however; the comparison between hour 12 and 2 yielded 9 DEGs. All except 1 were down-regulated at hour 12. The hour 12 vs. 0 comparison revealed 49 DEGs of which 24 were downregulated at hour 12. In conclusion, results revealed some pathways associated with energy metabolism that were altered in response to EHC, with most differences in the nCPa. Surprisingly, fewest differences were observed in the PVN. Findings provide some future target regions, such as the nCPa, and metabolic pathways to better understand how EHC affects stress responses and energy homeostasis later in life. eggs were kept at 26.6ºC for 12 hours and then were randomly divided into a control and embryonic heat conditioning (EHC) group. These groups were separated into two incubators (Rite Farm Products Pro-1056) and labeled control and EHC, respectively. Both groups from embryonic day (ED) 0-7 were incubated at 37.5ºC and 80% relative humidity. The eggs in the control group continued to be incubated at 37.5ºC until hatch at ED 21. The EHC group was separated from the control group at ED 7 and introduced to an increase in temperature to 39.5ºC and 80% relative humidity for 12 hours per day (07:30–19:30), and 37.5ºC for the remainder of the 24 hours until ED 16. The EHC group then spent the remaining 5 days at 37.5ºC until hatch. After ED 18.5, all eggs were subjected to candling. Infertile eggs and dead embryos were removed from the study. Eggs with viable embryos were transferred to a common hatching incubator (Rite Farm Products Pro-264) and maintained within their separate groups (separate enclosed trays to prevent mixing of treatments) at 36.9ºC and 50% relative humidity for 18 hours. The temperature was gradually decreased to 35ºC until the hatch was collected. Hatched chicks were arbitrarily divided into group cages based on treatment in preparation for the acute day 4 post-hatch heat challenge. The holding room temperature was set at 30ºC, with ad libitum access to food and water, and 24 hr continuous light exposure, similar to our previous studies. On day 4 post-hatch, Control and EHC groups were subjected to an acute heat challenge at 36ºC. Samples were collected at three timepoints: 0 (before heat challenge; baseline), 2 hours, and 12 hours from the start of the increased temperature. Within these three time points, 10 birds were collected and euthanized from control and EHC groups, with 20 birds total for each time point. Chicks were weighed individually, euthanized by decapitation, and sexed via gonad identification.